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Knockdown of CXCL5 inhibits the invasion, metastasis and stemness of bladder cancer lung metastatic cells by downregulating CD44.

ABSTRACT: In our previous studies, we found that T24 lung metastatic cancer cells showed high invasion and metastasis abilities and cancer stem cell characteristics compared with T24 primary cancer cells. By screening for the expression of CXC chemokines in both cell lines, we found that CXCL5 is highly expressed in T24-L cells. The aim of this study is to shed light on the relationship of CXCL5 with epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs). RNAi technology was used to decrease CXCL5 expression in the T24-L cell line, and the EMT and CSCs of the shCXCL5 group and the control group were compared. The CXCR2 inhibitor SB225002 was used to inhibit the receptor of CXCL5 to determine the effect of the CXCL5/CXCR2 axis. The knockdown of CXCL5 expression in T24-L cells reduced their EMT and CSC characteristics. RT-PCR and Western blot analyses revealed the downregulation of N-cadherin, Vimentin and CD44. In addition, when CD44 expression was knocked down, the EMT ability of the cells was also inhibited. This phenomenon was most pronounced when both CXCL5 and CD44 were knocked down. CXCL5 and CD44 can affect the EMT and stem cell capacity of T24-L cells through some interaction.

SUBMITTER: Wang W

PROVIDER: S-EPMC8670357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBladder carcinoma is one of the most common urological cancers. ITPR3, as a ubiquitous endoplasmic reticulum calcium channel protein, was reported to be involved in the development and progression of various types of cancer. However, the potential roles and molecular mechanism of ITPR3 in bladder cancer are still unclear. Herein, we elucidated a novel role of ITPR3 in regulating the proliferation, metastasis, and stemness of bladder cancer cells.MethodsThe expression of ITPR3 in bladder cancer was analyzed using public databases and bladder cancer tissue microarrays. To demonstrate the role of ITPR3 in regulating the NF-ĸB/CD44 pathway and the progression of bladder cancer, a series of molecular biology and biochemistry methods was performed on clinical tissues, along with in vivo and in vitro experiments. The methods used included western blot assay, quantitative RT-PCR assay, immunofluorescence assay, immunohistochemistry (IHC) assays, wound healing assay, Transwell assay, colony formation assay, tumorsphere formation assay, cell flow cytometry analysis, EdU assay, MTT assay, cell transfection, bisulfite sequencing PCR (BSP), a xenograft tumor model and a tail vein cancer metastasis model.ResultsHigher ITPR3 expression was found in bladder cancer tissues and bladder cancer cells compared with the corresponding normal peritumor tissues and SV-HUC-1 cells, which was attributed to demethylation in the ITPR3 promoter region. ITPR3 promoted the proliferation of bladder cancer by accelerating cell cycle transformation and promoted local invasion and distant metastasis by inducing epithelial-to-mesenchymal transition (EMT). Meanwhile, ITPR3 maintained the cancer stemness phenotype by regulating CD44 expression. NF-κB, which is upstream of CD44, also played a critical role in this process.ConclusionsOur study clarifies that ITPR3 serves as an oncogene in bladder cancer cells and represents a novel candidate for bladder cancer diagnosis and treatment.

Project description:Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods: CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O-glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O-glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics allowed, for the first time, the comprehensive characterization of CD44 splicing code at the protein level. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification as well as associated glycoforms. Finally, we confirmed the link between CD44 and invasion in CD44s-enriched cells in vitro by small interfering RNA (siRNA) knockdown, supporting findings from BC tissues. The key role played by short-chain O-glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Conclusions: Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/ Sialyl-Tn (STn) as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in health and disease.

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Knockdown of CXCL5 inhibits the invasion, metastasis and stemness of bladder cancer lung metastatic cells by downregulating CD44.

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